Transient gene expression in electroporated protoplasts and intact cells of sugar beet

Factors influencing the transient expression of introduced foreign DNA in electroporated protoplasts and intact cells of sugar beet were determined by assaying for the activity of chloramphenicol acetyltransferase (CAT), using a rectangular pulse generating system. Extractable CAT activity depended upon 1) applied plasmid DNA concentration, 2) protoplast density, 3) the interaction between pulse field strength, duration, number, time interval between pulses and the resultant effect on culture viability, and 4) the physiological state of the protoplasts. Mesophyll protoplasts were more susceptible to damage by electroporation, and were more specific in their requirement for electroporations which allowed CAT expression, than were protoplasts derived from suspension culture cells. CAT activity was also demonstrated, at low levels, after electroporation of intact suspension culture cells, and could be increased by pectinase treatment of the cells before electroporation.     

Direct gene transfer to plant protoplasts by mild sonication

Summary A novel procedure employing mild sonication for transformation of plant protoplasts is described. Transient expression of a chloramphenicol acetyltransferase (CAT) gene in protoplasts of sugar beet (Beta vulgaris L.) and tobacco (Nicotiana tabacum L.) was obtained by a brief exposure of the protoplasts to 20 kHz ultrasound in the presence of plasmid DNA. Maximum levels of CAT activity were achieved by sonication for 500–900 ms at 30–70 W electric power (0.65–1.6 W/cm2 acoustic power). This reduced the viability to 15–20 % and 60 % for sugar beet and tobacco protoplasts, respectively. Up to 12 % (sugar beet) and 81 % (tobacco) of maximum transient expression could be achieved with no significant loss of viability. Protoplasts surviving exposure to ultrasound were found to have a similar long-term viability and to regenerate to micro-calli as untreated protoplasts. Plasmid DNA concentrations of 80–110 g/ml and sucrose concentrations of 21–28 % in the sonication medium were found to be optimal for transient expression.Abbreviations CAT chloramphenicol acetyltransferase     

Gene transfer into isolated and cultured tobacco zygotes by a specially designed device for electroporation

 We have established a technique for isolating, culturing and transforming tobacco zygotes. Zygotes were isolated by microdissection or enzymatic maceration from fertilized embryo sacs. Viable zygotes cocultured with mesophyll protoplasts underwent first division after 3 days of culture. Zygotes isolated by microdissection underwent a higher frequency of first division (61.2%) than those isolated by enzymatic maceration (30.5%). Globular embryos were formed only from microdissected zygotes, at a frequency of 8.7% after 1–2 weeks in culture. An efficient millicell device for the electroporation of DNA into zygotes was established. The electroporated zygotes divided in vitro at a frequency of 54.6% and developed into proembryos. Introduced GFP gene constructs showed transient expression in about 2.6% of the electroporated tobacco zygotes. Received: 2 February 2000 / Revision received: 6 April 2000 / Accepted: 24 May 2000     

Comparative analysis of free DNA delivery and expression into protoplasts of Panicum maximum Jacq. (Guinea grass) by electroporation and polyethylene glycol

Relative levels of gene expression were studied in protoplasts isolated from two cell lines of Panicum maximum following DNA delivery by electroporation and polyethylene glycol (PEG). Gene expression was evaluated by assaying for chloramphenicol acetyltransferase (CAT) activity expressed by the CaMV 35S promoter with a nopaline synthase 3' polyadenylation signal, approximately 48 hours after DNA delivery. The expression of the CAT gene was slightly higher in electroporated protoplasts in comparison to PEG mediated delivery. However, PEG treated protoplasts showed higher plating efficiency. The effect of different salts and the molecular weight of PEG used on gene expression was also studied.     

Co-electroporation of rice protoplasts with RNAs of cucumber mosaic and tobacco mosaic viruses

Cucumber mosaic virus (CMV) RNA was used to study electroporation conditions suitable for protoplasts from rice suspension cultures. Rice protoplasts required a stronger and shorter electric pulse than tobacco protoplasts for introduction of viral RNA. Under optimized conditions, CMV infection was established in 65 % of electroporated protoplasts. In contrast, electroporation with tobacco mosaic virus (TMV) RNA did not result in infection of rice protoplasts. However, when TMV RNA was electroporated into rice protoplasts together with CMV RNA, TMV production was demonstrated in 15 % of protoplasts. Differential staining with fluorescent antibodies against the two viruses showed that the protoplasts producing TMV were without exception also infected by CMV. The results show that CMV replicates in rice protoplasts by itself, whereas TMV does so only with the aid of CMV.Abbreviations CMV cucumber mosaiv virus - PBS phosphate buffered saline - TMV tobacco mosaic virus.     

Genetic transformation of plants by protoplast electroporation

This article describes an optimized protocol for the electroporation of tobacco mesophyll protoplasts together with notes and data on the effects of various parameters and suggestions for work with protoplasts of other species. In this protocol, electroporation is achieved by means of electrical pulses from a high-voltage, capacitive-discharge unit. Procedures are described for measurement of protoplast viability with Evan's blue, the detection of transient expression of CAT and GUS gene plasmid constructs, and for the recovery of stable transformants based on selection for kanamycin resistance.     

Electroporation: Mechanism and transient expression, stable transformation and biological effects in plant protoplasts

The present state of knowledge about the mechanistic and theoretical aspects of electroporation is summarized. Parameters affecting the efficiency of transient expression and stable transformation of electroporated plant protoplasts are rewieved. Biological effects of electroporation on plant protoplasts are described.     

Visualizing mRNA expression in plant protoplasts: factors influencing efficient mRNA uptake and translation.

In this paper we demonstrate that RNA sequences present upstream and downstream of a reporter gene coding region play an important role in determining the amount of protein produced from an mRNA. A translational enhancer, omega, derived from tobacco mosaic virus, when present at the 5'-end of beta-glucuronidase mRNA increased the efficiency of translation 16-fold to 18-fold in electroporated tobacco or carrot protoplasts, and threefold to 11-fold in maize or rice protoplasts. The presence of omega did not alter the half-life of the mRNA in vivo. We also demonstrate for the first time that a minimum polyadenylated tail length of 25 adenylate residues is sufficient to substantially increase the expression and half-life of the reporter mRNA in plants. When in vitro-produced mRNAs were synthesized such that extra sequence was added to the 3'-end of the poly(A) tail, however, the final level of expression was decreased up to 80%. Omega, the translational enhancer, and a poly(A) tail function independently of each other; their combined effect on translation, when both are present in an mRNA, is the multiplication of their individual effects. Histochemical analysis for the presence of beta-glucuronidase in tobacco established that virtually all viable cells receive mRNA during electroporation. Video image analysis of tobacco protoplasts electroporated with luciferase mRNA demonstrated that there is a wide range in the level of expression of this marker. Carrier RNA, when present during electroporation, had only a modest effect on increasing mRNA uptake. Reporter mRNA expression in electroporated protoplasts was directly proportional to the input mRNA up to at least 30 micrograms/ml.     

Targeting of T7 RNA polymerase to tobacco nuclei mediated by an SV40 nuclear location signal

We have expressed two T7 RNA polymerase genes by electroporation into tobacco protoplasts. One of the genes was modified by inserting nucleotides encoding a viral nuclear localization signal (NLS) from the large T antigen of SV40. Both T7 RNA polymerase genes directed synthesis of a ca. 100 kDa protein in the electroporated protoplasts. T7 RNA polymerase activity was detected in extracts of protoplasts electroporated with both genes. Immunofluorescence analysis of these protoplasts indicated that only the polymerase carrying the NLS accumulated in the cell nucleus. These experiments suggest that mechanisms involved in the transport from the cytoplasm to the nucleus are similar in plant and animal cells. This system demonstrates the feasibility of T7 RNA polymerase-based approaches for the high-level expression of introduced genes in plant cells.     

Optimization of electroporation for transfection of mammalian cell lines

Electroporation can be a highly efficient method for introducing DNA molecules into cultured cells for transient expression of genes or for permanent genetic modification. However, effective transformation by electroporation requires careful optimization of electric field strength and pulse characteristics. We have used the transient expression of the firefly luciferase gene as a rapid and sensitive indicator of gene expression to describe the effects on transfection efficiency of altering electroporation field strength and shape. Using the luciferase assay, we investigated the correlation of cell viability with optimal transfection efficiency and determined the optimal parameters for a number of phenotypically distinct mammalian cell lines derived from the nervous and immune systems. The efficiency of electroporation under optimal conditions was compared with that obtained using DEAE-dextran or calcium phosphate-mediated transformation. Transfection by electroporation using square wave pulses, as opposed to exponentially decaying pulses, was found to be significantly increased by repetitive pulses. These methods improve the ability to obtain high efficiency gene transfer into many mammalian cell types.     

Introduction of impermeable molecules into pollen grains by electroporation

Summary Electroporation was used to introduce plasma membrane impermeable molecules into the cytoplasm of pollen grains ofLilium longiflorum. Ungerminated pollen grains were exposed to the fluorescent dye quin2 or FITC-labelled dextrans and electroporated with exponentially decaying voltage pulses of 250 to 2000 V/cm and time constants of 0.01 to 10 s. The number of electroporated pollen grains increased with the strength and duration of the voltage pulses, and with the osmolarity of the external medium. Optimal results were obtained with pulses of 1000 V/cm and 10 s time constant, and with 900 mM mannitol in the electroporation buffer. The size of the pores produced in the plasma membrane by electroporation allowed uptake of 40 kDa dextran but not 70 kDa dextran. The rate of germination of pollen grains was low immediately after electroporation, but increased with time in pollen growth medium. The conditions of electroporation reported here may be used to load genetic material into pollen grains for the production of transgenic plants.Abbreviations PGM pollen growth medium - FDA fluorescein diacetate - FITC fluorescein isothiocyanate     

Soybean protoplast culture and direct gene uptake and expression by cultured soybean protoplasts

A method was developed for culturing protoplasts freshly isolated from developing soybean (Glycine max L.) cotyledons. First cell divisions were observed within 5 days after protoplast isolation and microcalli, consisting of about 20 cells, were formed within 10 days. Thirty days after protoplast isolation, callus tissues were observed without the aid of a microscope. A 30 to 50% plating efficiency was consistently obtained. Using a polyethylene glycol-electroporation technique, DNA was introduced into these protoplasts. The protoplasts were then cultured to form callus. Chloramphenicol acetyltransferase (CAT) activity was detected in protoplast cultures 6 hours after introduction of a 35S-CAT-nopaline synthase 3′ chimeric gene. The highest CAT activity was detected in 3-day-old electroporated protoplast cultures, indicating transient expression of the introduced gene. Some CAT activity was detected in 40-day-old callus cultures and in geneticin (G418) selected callus tissues which also received a chimeric neomycin phosphotransferase II gene, indicating the presence of stable transformants. A control chimeric gene with an inverted 35S promoter failed to produce any CAT activity in this system.     

Experession and integration of genes introduced into highly synchronized plant protoplasts

Summary Chimaeric genes containing the chloramphenicol acetyltransferase (CAT) coding sequence were introduced into protoplasts of suspension-cultured tobacco cells using improved conditions of electroporation (Okada et al. 1986). CAT activity became detectable in the protoplasts within 3 h, was maximal during a period of 18–36 h after electroporation, and then declined gradually. Alpha-amanitin added to the medium abolished the transient expression of the CAT gene. The closed circular form of input DNA was as effective as the linear form for the transient expression. The suspension culture was treated with aphidicolin, and S, G₂, M and G₁ phases were identified in the highly synchronized cell cycle obtained by releasing the cells from the inhibition of DNA synthesis. When a chimacric CAT gene was introduced into M phase protoplasts prepared from the synchronized culture, the transient expression of the CAT gene was 3–4 times higher than when it was introduced into protoplasts of other cell cycle phases. The frequency of stable transformation with a chimaeric neomycin phosphotransferase II gene was studied using the same system. G-418-resistant transformants were obtained from M phase protoplasts at frequencies 2–8 times those obtained from protoplasts at other cell cycle phases. The results indicate that the absence of the nuclear membrane in mitotic cells favours delivery to the nucleus of exogenous DNA introduced into the cytoplasm.     

Direct gene transfer to protoplasts of two genotypes of Asparagus officinalis L. by electroporation

Callus-derived protoplasts of two genotypes of asparagus (Asparagus officinalis L.) were electroporated to introduce the -glucuronidase gene (GUS). The level of GUS transient gene expression and the viability of the protoplasts were influenced by the voltage and duration of the electric pulse. The transient expression level was enhanced by increasing the plasmid DNA concentration and by the presence of polyethylene glycol (PEG) in the electroporation medium. A considerable increase in GUS activity was observed in presence of both PEG and upon heat-shock treatments compared to PEG treatment alone. An optimal level of GUS activity was obtained after electroporation with a capacitive discharge of 500 V/cm and 94 ms duration in both genotypes. The two genotypes differed in their responses to in vitro culture and also showed variable levels of transient expression. The present technique was found to be suitable to obtain transgenic plants as the histochemical GUS assay revealed GUS activity in the protoplast-derived micro-colonies as well as in callus tissues.Abbreviations CaMV cauliflower mosaic virus - CAT chloramphenicol acetyltransferase - CPW salts Cocking-Power-White salts - MES 2-[N-Morpholino]ethanesulfonic acid - MU 4-methyl umbelliferone - MUG 4-methyl umbelliferyl glucuronide - F microfarad - NAA naphthaleneacetic acid - NOS nopaline synthase - NPT II neomycin phosphotransferase - X-Gluc 5-bromo-4-chloro-3-indolyl--D-glucuronide     

Effects of electroporation pulse wave on the incorporation of viral RNA into tobacco protoplasts

The uptake and expression of cucumber mosaic viral (CMV) RNA by tobacco protoplasts was examined using both square wave and exponential wave electroporation pulses. These electropulses, when supplied at sufficient field strength for a critical duration, enabled RNA to be incorporated and expressed in more than 60% of the surviving protoplasts. The results of experiments using these two electroporation wave forms showed significant differences in RNA uptake and expression. The number of viable protoplasts and cells showing expression of RNA was higher over a much broader range of experimental conditions using the square rather than exponential wave generator, even when both machines were optimized for maximal performance. However, at a narrow range of low field strengths the exponential wave pulse generated a higher percentage of transformants than did the square wave pulse. It was shown that after an electroporation pulse from either wave form, there were viable cells which expressed foreign RNA at predictable levels.     

Transformation of sweet potato tissues with green-fluorescent protein gene

Summary The expression of the green-fluorescent protein (GFP) gene from Aequorea victoria (jellyfish) was analyzed by transient and stable expression in sweet potato Ipomoea batatas L. (Lam.) ev. Beauregard tissues by electroporation and particle bombardment. Leaf and petiole segments from in vitro-raised young plantlets were used for protoplast isolation and electroporation. Embyrogenic callus was also produced from leaf segments for particle bombardment experiments. A buffer solution containing 1×10⁶ protoplasts ml⁻¹ was mixed with plasmid DNA containing the GFP gene, and electroporated at 375 V cm⁻¹. Approximately 25–30% of electroporated mesophyll cell protoplasts subsequently cultured in KM8P medium regenerated cell walls after 48 h. Of these, 3% emitted bright green fluorescence when exposed to UV-blue light at 395 nm. Transformed cells continued to grow after embedding in KM8P medium solidifed with 1.2% SeaPlaque agarose. Stable expression of GFP was observed after 4 wk of culture in approximately 1.0% of the initial GFP positive cells (27.5 GFP positive micro callases out of 3024 cells which transiently expressed GFP 48 h after electroporation). In a separate experiment, 600–700 bright green spots were observed per plate 48 h after bombarding leaf segments or embryogenic cellus. In bombarded cultures, several stable GEP-expressing sectors were observed in leafderived embryogenic callus grown without selection for 4 wk. These results show that GFP gene expression can occur in various sweet potato tissues, and that it may be a useful sereenable marker to improve transformation efficiency and obtain transgenic sweet potato plants.     

Heat Inducible Expression of a Chimeric Maize hsp70CAT Gene in Maize Protoplasts

The response of maize (Zea mays L.) protoplasts to high temperature stress was investigated. After isolation and electroporation, protoplasts were preincubated for 12 hours at 26°C then incubated for 6 hours at elevated temperatures. The pattern of polypeptides synthesized by these protoplasts during the last hour was monitored by in vivo labeling with ³⁵S-methionine. Incubation at 40° and 42°C resulted in the synthesis of polypeptides not detectable at 26°C. Introduction of a chimeric maize heat shock protein 70 promoter-chloramphenicol acetyltransferase coding region gene into protoplasts via electroporation resulted in the temperature-dependent induction of chloramphenicol acetyltransferase activity with maximal activity at 40°C. In the same protoplasts, a second chimeric gene, in which the firefly luciferase coding region was under the control of the 35S promoter from cauliflower mosaic virus, did not show an increase in expression after incubation at higher temperatures. Maize protoplasts provide a system to study molecular responses to high temperature stress.     

Introduction and expression of foreign DNA in isolated spinach chloroplasts by electroporation

An electroporation-mediated method for the study of foreign gene expression within chloroplasts has been developed. The chloroplast expression vector pHD203-GUS, which consists of coding regions for β-glucuronidase (GUS) and chloramphenicol acetyltransferase (CAT) separated by a double psbA promoter fragment from pea (in opposite orientation) was electroporated into spinach chloroplasts and the transient gene expression was examined. Conditions for the expression of the reporter genes have been optimized. Both CAT and GUS activities were detected in chloroplasts electroporated with pHD203-GUS, but not with nuclear expression vector pBI221 or negative control pUC18. No GUS activity was detected when pHD203-GUS was electroporated into spinach protoplasts. Dot immunoblot analysis using anti-GUS antibody confirmed the existence of GUS protein in chloroplasts electroporated with chloroplast-specific vector but not the negative controls, excluding the possibilities of endogenous GUS or bacterial contamination. The expression of GUS protein in treated chloroplasts was further confirmed by Western blot analysis.     

Transient expression of electroporated DNA in monocotyledonous and dicotyledonous species

Transient expression of electroporated DNA was monitored in protoplasts of several monocot and dicot species by assaying for expression of chimeric chloramphenicol acetyltransferase (CAT) gene constructions. Expression was obtained in the dicot species of Daucus carota, Glycine max and Petunia hybrida and the monocot species of Triticum monococcum, Pennisetum purpureum, Panicum maximum, Saccharum officinarum, and a double cross, trispecific hybrid between Pennisetum purpureum, P. americanum, and P. squamulatum. Recovery and viability of protoplasts after electroporation decreased with increasing voltages and capacitance while CAT activity increased up to a critical combination of voltage and capacitance beyond which the activity dramatically decreased. The optimal compromise between DNA uptake and expression versus cell survival was determined for D. carota and applied successfully to the other species. Maximum transient expression occurred 36 hours after electroporation of D. carota. The potential for using this procedure to rapidly assay gene function in dicot and monocot cells and application of this technique to obtain transformed cereals is discussed.     

Optimized conditions for transgenesis of the ascidian Ciona using square wave electroporation

The protochordate Ciona has recently become an important model organism for the study of developmental gene regulation, in part because transient transgenic embryos can be produced rapidly and reliably using electroporation. Published methods are currently for the use of electroporation devices delivering exponentially decaying pulses. However, some workers have advocated the use of square wave electroporation devices for eukaryotic transgenics. This paper presents results of experiments to find optimal conditions for the use of square-wave electroporation in the ascidian Ciona. In the present analysis, a single pulse of 90 msec duration at 63–75 V/cm was found to give an optimal balance of a high proportion of cells transformed with the transgene, and a low level of abnormal development. Forty micrograms of DNA per electroporation is sufficient for effective visualization of reporter gene expression, although doses up to 100 μg provide higher proportions of transformed cells. In side-by-side comparison with exponential pulse electroporation, square wave pulses give similar penetrance of transgene expression, along with lower proportions of embryos with abnormal development at higher amounts of input DNA.